Malignant melanoma is a serious health issue for Veterans, and metastatic melanoma is usually incurable if it metastasizes to distant sites. Exciting and impressive data with immune checkpoint blockade demonstrate the ability of the immune system to produce durable responses in some metastatic melanoma patients and have changed the standard of care1. Effective treatment with immune checkpoint blockade seems to require activation of anti-melanoma T cells that are specific for a wide variety of melanoma antigens including patient- unique neoantigens. Canine malignant melanoma provides an excellent preclinical model to study melanoma immunotherapy as it is similar to human melanoma occurring spontaneously in the setting of an intact immune system and with metastasis occurring via lymphatics or blood vessels to regional lymph nodes, lungs, liver, brain, and kidney. Germane to this application, the GD2 disialoganglioside (GD2) is expressed in both human and canine melanoma2-4. We therefore propose intratumoral (IT) injection of the GD2-reactive hu14.18-IL2 immunocytokine (IC) (IT-IC) alone, and in combination with other therapies synergistic in preclinical murine models, in companion dogs with melanoma to convert the injected tumor into an effective in situ tumor vaccine5. Our central hypothesis is that IT-IC in combination with local radiation therapy (RT) in canine melanoma can induce a T cell response to melanoma. Further, we hypothesize that this response can be amplified with immune checkpoint blockade, and that this regimen is safe and well tolerated. These hypotheses will be tested by achieving the following: Aim 1) Determine toxicity, immunogenicity, and assess antitumor activity of IT delivery of hu14.18-IL2 alone and with local RT in dogs with locally advanced or metastatic melanoma; Aim 2) Evaluate local and systemic antitumor activity following IT delivery of hu14.18- IL2 combined with RT and immune checkpoint blockade with anti-PD1 in dogs with locally advanced or metastatic melanoma; and Aim 3) Identify biomarkers to inform combination immunotherapy strategies with IT delivery of hu14.18-IL2 in dogs with locally advanced or metastatic melanoma. Standard clinical assessments of toxicity and tolerance, as well as measurement and imaging of clinically evident disease, are part of Aims 1 and 2. Serial blood samples and tumor biopsies will allow for detailed histologic and immunologic assessments in Aim 3 to determine mechanisms of antitumor activity and to determine whether histologic findings of concomitant immune tolerance seen in our murine model are also present in the dog. Exploratory lab studies will: i) evaluate alterations in the cellular milieu in the periphery and tumor before and after immunotherapy, ii) utilize novel immune monitoring to identify a candidate biomarker of response for dogs with melanoma receiving IT-IC, and iii) assess the diversity and clonality of intratumoral and peripheral blood T cell receptor repertoire. This study uses spontaneous canine melanoma to test a novel RT + IT-IC + immune checkpoint blockade regimen already proven to induce long lasting, potent and effective elimination of large local and distant established melanoma lesions in tumor-bearing mice5. The results from this canine trial could enable rapid translation into clinical testing of these concepts in human patients, including Veterans, with advanced melanoma. This approach is clinically advantageous as it involves agents/treatments that are currently available or in clinical testing, and it could be readily made available as an ?off the shelf? therapy in Veterans Hospitals throughout the USA. Moreover, it personalizes treatment by targeting potent and patient- specific neoantigens. Finally, RT + IT-IC + immune checkpoint blockade could be incorporated into treatment of virtually any other malignancy, including other common malignancies in Veterans. The only requirement for this strategy is having a tumor-reactive monoclonal antibody (mAb) for the malignancy being targeted, analogous to hu14.18, that could either be formulated as an IC or for which an IC exists.